Antibody persistence of standard versus double three-dose hepatitis B vaccine in liver transplant children: a randomized controlled trial

Rapid hepatitis B (HB) surface antibody (anti-HBs) loss is prevalent after liver transplantation (LT). Herein, we evaluated anti-HBs persistence after HB vaccination using two regimens in LT children. We recruited 66 previously immunized LT children with anti-HBs level of < 100 mIU/mL. Participants were randomly reimmunized with standard-three-dose (SD) and double-three-dose (DD) intramuscular HB vaccination at 0, 1, and 6 months. Anti-HBs were assessed at every outpatient visit. Antibody loss defined as anti-HBs levels < 100 mIU/mL after three-dose vaccination. After three-dose vaccination, 81.8% and 78.7% of participants in the SD and DD groups, had anti-HBs levels > 100 mIU/mL, with a geometric mean titer (GMT) of 601.68 and 668.01 mIU/mL (P = 0.983). After a mean follow-up of 2.31 years, the anti-HBs GMT was 209.81 and 212.61 mIU/mL in the SD and DD groups (P = 0.969). The number of immunosuppressants used and an anti-HBs level < 1 mIU/mL at baseline were independently associated with anti-HB loss. The DD regimen strongly increased the risk of anti-HBs loss (adjusted hazard ratio, 2.97 [1.21–7.31]; P = 0.018). The SD HB reimmunization regimen effectively maintained protective anti-HBs levels in children undergoing LT, making it the preferred regimen for such children with anti-HB loss. Trial registration: TCTR20180723002.


Statistical analysis
Continuous data are presented as mean (standard deviation) or median (interquartile range) values based on the distribution of variables.Categorical data are presented as numbers and percentages.The unpaired t-test and Mann-Whitney U test were used to compare the data, as appropriate.Discrete data were compared using the chi-square test or Fisher's exact test, as appropriate.The geometric mean titer (GMT) was calculated based on an anti-HBs titer of > 1 mIU/mL and represented logarithmically.The failure rate or anti-HB loss rate was

Cox regression analysis of factors associated with anti-HBs loss
Univariate Cox regression analysis revealed an association between anti-HBs loss over time, age at vaccination, and time from LT to vaccination (Table 3).The analysis also supported an increased risk of anti-HB loss over time in children who had undergone LT and received multiple immunosuppressants (IMs).Multivariate Cox regression analysis revealed that the number of IMs used, anti-HBs level < 1 mIU/mL before vaccination, and double-dose vaccination (Table 3) were associated with anti-HB loss with time.The number of IMs was strongly associated with an increased risk of anti-HBs loss over time.Administration of two and three IMs had adjusted hazard ratios (HRs) of 4.86 (95% CI 1.64-14.45;P = 0.004) and 17.65 (9%% CI 3.96-78.68;P < 0.001), respectively.An anti-HBs level of < 0.1 mIU/mL before vaccination was strongly associated with an increased risk of anti-HB loss over time (HR, 3.85 [1.08-13.65]).Participants in the DD group also had an increased risk of anti-HBs loss over time (HR, 2.97 [1.21-7.31];P = 0.018) (Fig. 3).

Discussion
This study demonstrated the efficacy of SD and DD HB vaccination during long-term follow-up in children who had undergone LT.In the SD and DD groups, 60.4% and 41% of participants, respectively, had persistent protective levels of anti-HBs (> 100 mIU/mL) at the 4-year follow-up, respectively.Multivariate Cox regression analysis showed that among children who had undergone LT, the use of multiple IMs and an anti-HBs level of < 1 mIU/ mL before vaccination were strongly associated with an increased risk of anti-HB loss over time.In addition, the DD regimen was an independent factor that increased the risk of anti-HBs loss over time.
To the best of our knowledge, the present study is the first RCT to compare the effectiveness of the SD and DD vaccines (at 0, 1, and 6 months) in previously immunized children who had undergone LT and experienced anti-HB loss.Our previous study, which had a short-term follow-up duration, demonstrated that complete SD or DD HB vaccination helped maintain a higher anti-HB level than after a booster HB vaccine dose 25 .The antibody response after completion of the DD HB vaccine regimen tended to be lower than that after completion of the SD regimen in our previous study.Similarly, the multivariate Cox analysis in the present continuous study revealed that among children who had undergone LT, the DD HB vaccine regimen carried a higher risk of anti-HBs loss over time.
Vaccination is the most effective interventional therapy for controlling infectious diseases.However, vaccine effectiveness is adversely affected by repeated vaccinations [26][27][28] , immune imprinting 29,30 , pre-existing anti-vector immunity 31 , short intervals between repeated vaccines 32,33 , and the co-administration of multiple vaccines 34 .Immune interference is the common phenomenon in influenza vaccine and coronavirus disease 2019 vaccine because of their variable antigenic sites in viral proteins.Immune imprinting of previous vaccinations and mismatching between viral strains and vaccine strains are the explanation of the vaccine ineffectiveness with the repeated vaccination 35 .Unlike that for the influenza and the coronavirus disease 2019 vaccines, there is limited evidence supporting immune interference with the HB vaccine.Fonzo et al. demonstrated that delaying HB vaccine administration within the first year of life could affect the long-term maintenance of anti-HBs levels.Each month of delay within the first year of life was associated with a 16% reduction in maintaining an anti-HB titer of < 10 IU/mL approximately 20 years after primary vaccination 36 .Impaired T-cell function and the fewer interactions between B and T cells in infants 37,38 necessitate repeated antigenic administrations, and a longer interval between repeat vaccine administration effectively maintains the appropriate anti-HBs level.However, the mechanism behind the antigenic overload that might interfere with the immune response to the HB vaccine has not been documented so far.Double dose regimen for hepatitis B vaccine was effective in HIV-infected patients as higher immunogenicity was observed, when it was measured 4-6 weeks and > 12 months after completion of the vaccination compared with standard dose regimen 20 .The conflict result of the anti-HBs response to double dose HB vaccine regimen from the present study and the study in HIV-infected patients might be explained by www.nature.com/scientificreports/ the complexity of cellular immune interactions after revaccination at higher doses in different immunocompromised patients that requires further investigation.This study also identified the use of multiple IMs and anti-HB levels of < 1 mIU/mL before revaccination as other factors strongly associated with anti-HB loss over time.Although B cells are key mediators of rigorous immunity that prevent HB infection, T cells play a vital role in stimulation B-cells for antibody production 39 .HIV positivity and a low CD4 count are examples of T cell defects leading to poor anti-HB response and long-term persistent immunity after revaccination 40 .In our previous study, using an enzyme-linked immunosorbent spot assay to evaluate the cellular immune response to the HB vaccine, we demonstrated a significant T-helper1 cell response with significantly higher-secreting cells in responders than in hyporesponders 25 , which was in line with the findings of Ni et al. 13 .In addition to T and B cell responses after vaccination, immune memory should be sufficiently rigorous to protect against pathogens.Ample evidence shows waning immunity with a rapid anamnestic response in healthy individuals with an anti-HBs level of < 10 mIU/mL, do not require revaccination 6 whereas anti-HBs loss might reflect immunity loss as evidenced by DNH in LT children.The present study found that the cutoff anti-HBs level of < 1 mIU/mL was significantly associated with the rate of anti-HB loss over time after HB vaccination.The low quantity or quality of immune memory cells in participants with very low anti-HBs levels might explain the increased risk of anti-HBs loss over time.This merits further investigation.In terms of clinical implications, while the number of IMs required depends on the patient's condition after LT and may not be modified, increasing anti-HB levels before revaccination after LT could be improved by administering a booster dose to children waiting for LT.Generally, the anti-HBs level will decrease with time after LT, but a booster dose of HB vaccination before LT would ensure that the anti-HBs level remains adequate to prevent DNH during 6-month post-LT.After that, monitoring anti-HBs level and revaccination with 3-dose HB vaccine when anti-HBs level declines to less than 100 mIU/mL.We suggest that this strategy be used for increasing and maintaining a high anti-HBs level during long-term follow-up to guarantee protection against HB infection in children who undergo LT.
A limitation of this study is that we included only a few cases of LT with anti-HBc-positive liver grafts (n = 2).Such patients have a high risk of DNH, and this requires a separate subgroup evaluation, as their immune response after vaccination might differ from that of children who undergo LT with anti-HBc-negative liver grafts.Furthermore, the role of the cellular immune response in the persistence of anti-HBs during long-term followup was not evaluated in this study.Last, the power of this study is low because of the small number of recruited participants that might lead to statistical insignificance of the main primary outcome or the rate of anti-HBs loss overtime comparing between SD and DD regimen.However, according to multivariate Cox regression analysis that included the clinically significant parameters in the data analysis, we found that DD was an independent Table 2. Anti-HBs persistence rate (anti-HBs level > 100 mIU/mL) in the standard and double dose groups.Anti-HBs hepatitis B surface antibody, CI confidence interval.

Figure 1 .
Figure 1.Analysis population and patient flow.

Figure 2 .
Figure 2. Comparison of estimated anti-HBs persistence rates between the standard and double-dose groups.

Table 1 .
Patient demographics and characteristics at baseline by vaccination arm.*P-values obtained using the Mann-Whitney U and Chi-square tests.Anti-HBc hepatitis B core antibody, Anti-HBs hepatitis B surface antibody, GGT gamma glutamyl transpeptidase, GMT geometric mean titer, HB hepatitis B, Hb Hemoglobin, AST serum aspartate aminotransferase, ALT serum alanine aminotransferase, LT liver transplant, WBC white blood cell count.